Ryanodine Receptor Ca(2+) release channels (RyRs) play a key role in regulation of intracellular [Ca(2+)] during the process of Excitation-Contraction Coupling (EC Coupling). Full understanding of how the intracellular Ca(2+) is regulated is one of the fundamental goals of muscle research. It has been proposed that the domain-domain interactions within RyR are involved in stabilizing certain conformational states of the channel and could serve as the determining factors for the ability of certain RyR ligands to modulate Ca(2+) release. Identification and characterization of these molecular determinants of Ca(2+) release are the necessary steps for elucidating the mechanisms underlying intracellular Ca(2+) homeostasis. Work funded by this proposal will utilize molecular biological techniques to determine the regions of the RyR that interact with the central region of the channel, previously proposed to involved in domain-domain interactions. The role of these interactions in regulations of Ca(2+) release will be determined by monitoring macroscopic Ca(2+) transient using video rate light and confocal microscopy of RyR deficient myogenic cells expressing RyRs with mutations that are predicted to disrupt domain-domain interactions. This work will provide novel information needed to better understand the process of EC coupling and also may shed new light on the nature of muscle disorders such as malignant hyperthermia.